rabbit anti-cdk6, cell signaling  (Cell Signaling Technology Inc)

Journal: eLife

Article Title: Kinases in motion: impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/elife.94755

Figure Lengend Snippet: Figure 4. CDK4/6 interactions and conformations. (A) Illustration of regulatory CDK4/6 interactions and Rb activation. (B) Domain organization of CDK4, CDK6, and p16INK4a; tested point mutations are listed. (C) 3D structure of CDK6 in complex with p16INK4a. Crucial amino acids involved in the interaction of the two proteins are highlighted. The R31C mutant is depicted in orange (PDB code: 1BI7, Russo et al., 1998). (D) Protein:protein interaction (PPI) reporter analyses of the kinases CDK4 and CDK6 with p16INK4a. Scheme illustrates CDK4/6 hetero-dimer formation with p16INK4a analyzed using a PCA RLuc PPI reporter system. PPI induces the complementation of RLuc PCA fragments promoting an increase in bioluminescence (HEK293T cells, 48 hr of transient reporter expression). Bars represent the RLU fold change of PPI in relation to wild-type CDK4/6:p16INK4a complex (mean ± SEM, n=7 ind. experiments). (E) Basal signal of CDK4/6 KinCon reporters with indicated mutations are shown (expressed for 48 hr in HEK293T cells) (mean

Article Snippet: DOI: https://doi.org/10.7554/eLife.94755 20 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct mouse pcDNA3.1CDK6- F[1] CDK6- PPI reporter Transfected construct mouse pcDNA3.1CDK6- R31C- F[1] CDK6- PPI reporter (R31C) Transfected construct mouse pcDNA3.1CDK4- F[1] CDK4- PPI reporter Transfected construct mouse pcDNA3.1CDK4- R24C- F[1] CDK4- PPI reporter (R24C) Transfected construct mouse pcDNA3.1p16INK4a- F[2] p16INK4a- PPI reporter Transfected construct mouse pcDNA3.1- p16INK4a- P40L- F[2] p16INK4a- PPI reporter (P40L) Transfected construct human pcDNA3.1F[1]-MEK1- F[2] MEK1- KinCon Transfected construct human pcDNA3.1- F[1]-MEK1- K57E- F[2] MEK- KinCon (K57E) Transfected construct human pcDNA3.1F[1]-PKAc- F[2] PKAc- KinCon Transfected construct human pcDNA3.1F[1]-PKAc- L206R- F[2] PKAc- KinCon (L206R) Transfected construct human pcDNA3.1MO25- 3xFlag MO25- 3xFlag Transfected construct human pcDNA3.1F[1]-BRAF- F[2] BRAF- KinCon Transfected construct human pcDNA3.1F[1]-BRAF- V600E- F[2] BRAF- KinCon (V600E) Transfected construct human pcDNA3.1F[1]-RIPK1- F[2] RIPK1- KinCon Transfected construct human pcDNA3.1F[1]-RIPK2- F[2] RIPK2- KinCon Transfected construct human pcDNA3.1F[1]-RIPK3- F[2] RIPK3- KinCon Antibody GAPDH Rabbit monoclonal Cell Signaling 2118 1:10000 Antibody AMPKα Rabbit monoclonal Cell Signaling 2532 1:1000 Antibody PhosphoAMPKα(Thr172) Rabbit monoclonal Cell Signaling 2535 1:1000 Antibody LKB1 Rabbit monoclonal Cell Signaling 3047 1:1000 Antibody Vinculin Rabbit monoclonal Cell Signaling 4650 1:1000 Antibody RIP XP Rabbit monoclonal Cell Signaling 3493 1:1000 Antibody RIP3 Rabbit monoclonal Cell Signaling 13526 1:1000 Antibody CDK6 Mouse monoclonal Cell Signaling 3136 1:1000 Continued Continued on next page Kugler, Schwaighofer et al. eLife 2024;13:RP94755.

Techniques: Activation Assay, Mutagenesis, Expressing

Journal: eLife

Article Title: Kinases in motion: impact of protein and small molecule interactions on kinase conformations

doi: 10.7554/elife.94755

Figure Lengend Snippet: Figure 5. Impact of small molecules and protein interactions on kinase activity conformations. (A+B) Depiction of molecular interactions of a type I 1/2 and type III kinase inhibitors with a kinase domain (N and C lobe). Impact of PLX8394, Cobimetinib and GSK547 on wild-type (wt) and mutated versions of BRAF, RIPK1, and MEK1 kinase conformation (KinCon) reporters. 48 hr post transfection HEK293T cells expressing respective reporter constructs were treated with indicated inhibitors for 1 hr (1 μM) followed by RLuc PCA analyses (mean ± SEM, n=4/5 ind. experiments). (C) Depiction of molecular interactions of a type I kinase inhibitor with a kinase domain (N and C lobe). Impact of Abemaciclib on indicated CDK6 kinase conformations (wt and p16INK4a binding deficient). 48 hr post transfection HEK293T cells expressing respective reporter constructs were treated with indicated inhibitors for 3 hr (1 μM) followed by RLuc PCA analyses (mean ± SEM, n=4 ind. experiments). (D) Bioluminescence measurement of PKAc wt and L206R KinCon reporters. HEK293T cells expressing the reporter were treated with 20 μM of Forskolin for 15 min followed by RLuc PCA analyses (mean ± SEM, n=4 ind. experiments). (E) Kinome tree displays kinases for which KinCon reporters have been generated (red dots). The blue squares highlight the kinases for

Article Snippet: DOI: https://doi.org/10.7554/eLife.94755 20 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct mouse pcDNA3.1CDK6- F[1] CDK6- PPI reporter Transfected construct mouse pcDNA3.1CDK6- R31C- F[1] CDK6- PPI reporter (R31C) Transfected construct mouse pcDNA3.1CDK4- F[1] CDK4- PPI reporter Transfected construct mouse pcDNA3.1CDK4- R24C- F[1] CDK4- PPI reporter (R24C) Transfected construct mouse pcDNA3.1p16INK4a- F[2] p16INK4a- PPI reporter Transfected construct mouse pcDNA3.1- p16INK4a- P40L- F[2] p16INK4a- PPI reporter (P40L) Transfected construct human pcDNA3.1F[1]-MEK1- F[2] MEK1- KinCon Transfected construct human pcDNA3.1- F[1]-MEK1- K57E- F[2] MEK- KinCon (K57E) Transfected construct human pcDNA3.1F[1]-PKAc- F[2] PKAc- KinCon Transfected construct human pcDNA3.1F[1]-PKAc- L206R- F[2] PKAc- KinCon (L206R) Transfected construct human pcDNA3.1MO25- 3xFlag MO25- 3xFlag Transfected construct human pcDNA3.1F[1]-BRAF- F[2] BRAF- KinCon Transfected construct human pcDNA3.1F[1]-BRAF- V600E- F[2] BRAF- KinCon (V600E) Transfected construct human pcDNA3.1F[1]-RIPK1- F[2] RIPK1- KinCon Transfected construct human pcDNA3.1F[1]-RIPK2- F[2] RIPK2- KinCon Transfected construct human pcDNA3.1F[1]-RIPK3- F[2] RIPK3- KinCon Antibody GAPDH Rabbit monoclonal Cell Signaling 2118 1:10000 Antibody AMPKα Rabbit monoclonal Cell Signaling 2532 1:1000 Antibody PhosphoAMPKα(Thr172) Rabbit monoclonal Cell Signaling 2535 1:1000 Antibody LKB1 Rabbit monoclonal Cell Signaling 3047 1:1000 Antibody Vinculin Rabbit monoclonal Cell Signaling 4650 1:1000 Antibody RIP XP Rabbit monoclonal Cell Signaling 3493 1:1000 Antibody RIP3 Rabbit monoclonal Cell Signaling 13526 1:1000 Antibody CDK6 Mouse monoclonal Cell Signaling 3136 1:1000 Continued Continued on next page Kugler, Schwaighofer et al. eLife 2024;13:RP94755.

Techniques: Activity Assay, Transfection, Expressing, Construct, Binding Assay, Generated